Many of the actions of growth factors on cells are thought to be mediated by a family of inositol phosphoglycan (IPG) second messengers (T W Rademacher at al, Brazilian J. Med. Biol. Res., 27, 327-341, (1994)). It is thought that the source of IPGs is a "free" form of glycosyl phosphatidylinositol (GPI) situated in cell membranes. IPGs are thought to be released by the action of phosphatidylinositol-specific phospholipases following ligation of growth factor to receptors on the cell surface.
There is evidence that IPGs mediate the action of a large number of growth factors including insulin, nerve growth factor, hepatocyte growth factor, insulin-like growth factor I (IGF-I), fibroblast growth factor, transforming growth factor .beta., the action of IL-2 on B-cells and T-cells, ACTH signaling of adrenocortical cells, IgE, FSH and hCG stimulation of granulosa cells, thyrotropin stimulation of thyroid cells, cell proliferation in the early developing ear and rat mammary gland. However, to date, most of the research in this area has concentrated on the second messengers released by cells in response to insulin. For example, insulin stimulates rapid hydrolysis of membrane-associated GPI molecules in myocytes, adipocytes, hepatoma cells and T-cells. Recently, it has become clear that, at least where insulin is concerned, the released IPGs play an essential role as second messengers, and can in fact mimic many of the effects of insulin in the absence of the hormone.
Soluble IPG fractions have been obtained from a variety of animal tissues including rat tissues (liver, kidney, muscle brain, adipose, heart) and bovine liver. IPG biological activity has also been detected in malaria parasitized RBC and mycobacteria. The ability of an anti-inositolglycan antibody to inhibit insulin action on human placental cytotrophoblasts and BC3H1 myocytes or bovine-derived IPG action on rat diaphragm and chick ganglia suggests cross-species conservation of some three-dimensional features. However, it is well established that species-specific glycoconjugates are a common characteristic and structural characteristics determined on non-human derived IPG may not be found on the human derived material.
We have divided the family of IPG second messengers into distinct A and P-type subfamilies on the basis of their biological activities. In the rat, release of the A and P-type mediators has been shown to be tissue-specific (Kunjara et al, Biopolymers and Bioproducts: Structure, Function and Applications, J. Svast et al (ed), Dokya Publications, 301-306, (1995)). Although in the past it has not been possible to isolate single purified components from the tissue derived IPG fractions, much less in sufficient quantities to allow structural characterisation, there have been studies of the biological activities of the IPG containing fractions, and speculation as to the identity of the active components from non-human sources of the fractions based on indirect evidence from metabolic labelling and cleavage techniques.
Biological activity studies have shown that A-type mediators modulate the activity of a number of insulin-dependent metabolic effects such as acetylCoA carboxylase (activates), cAMP dependent protein kinase (inhibits), adenylate cyclase (inhibits) and cAMP phosphodiesterases (stimulates). In contrast, P-type mediators modulate the activity of enzymes such as pyruvate dehydrogenase phosphatase (stimulates) and glycogen synthase phosphatase (stimulates). The A-type mediators mimic the lipogenic activity of insulin on adipocytes, whereas the P-type mediators mimic the glycogenic activity of insulin on muscle. Both A and P-type mediators are mitogenic when added to fibroblasts in serum free media. The ability of the mediators to stimulate fibroblast proliferation is enhanced if the cells are transfected with the EGF-receptor. A-type mediators can stimulate cell proliferation in chick cochleovestibular ganglia.
Despite these studies, evidence for the presence of a family of soluble IPG-type mediators in a primary target organ for insulin action in humans has not yet been established. Further, research in this area has been severely hampered by the limited availability of the A and P-type IPGs in fractions derived from mammalian tissues. In particular, there have been experimental difficulties in identifying, isolating and characterising the active components of the IPG fractions having A- and P-type biological activity.
Thus, studies on the measurement in urine of chiro and myo inositol have been complicated by the fact that both breakdown of endogenous IPGs and dietary sources of the sugars will be present. Accordingly, prior art studies in this area which assumed that the P-type mediator contains chiro-inositol and that the A-type mediator contains myo-inositol must be interpreted with caution, see Fonteles, M C, Huang, L C, Larner, J, Diabetologia, 39:731-734, (1996), in which the authors report that they incorrectly identified the inositol in the P-type mediator which is pinitol and not chiro-inositol. As pinitol is not converted to chiro-inositol by the acid conditions used in carbohydrate analysis, this is a case of misidentification.
Further, analysis of material isolated by metabolic labelling with radionuclides or post-isolation labelling of extracted material cannot be related to the chemically active substance, since one is only following the labelled material and the actual active substance could co-isolate but not be labelled. In addition, various enzymic or chemical treatments of the compounds used two determine structural characteristics inactivate the compound making further structural steps impossible since one can no longer relate activity and structure. Further, as the active components of the A- and P-type IPG fractions are believed to be carbohydrates rather than proteins, they cannot be produced by recombinant DNA technology.
Thus, while there has been speculation in the art as to the chemical identity of these components, to date, there has been no isolation of an active component and no demonstration that it has A- or P-type biological activity.